1. Field of the Invention
This invention relates to an improved multilayer analysis film suitable for highly sensitive, quantitative analysis of transaminase activity.
2. Development of the Invention
Various transaminases are known as enzymes capable of transferring an amino group. Among these glutamate-pyruvate transaminase (GPT) and glutamate-oxaloacetate transaminase (GOT) blood concentrations may be indicative of liver diseases and, therefore, quantitative analysis of GPT and GOT activities is very important in the diagnosis of liver diseases.
GPT and GOT are transaminases which catalyze transamination between an amino acid and an .alpha.-keto acid to produce another amino acid, as shown by the following reaction schemes (1) and (2). ##STR1##
Enzyme assay may be classified into two methods; one is called an endpoint method for assaying an amount of substrate in which an enzyme-catalyzed reaction is quantitatively proceeded and after completion of the reaction, an amount of substrate or product changed is measured at the endpoint as a detectable species. The endpoint method thus involves a reaction system in which a detectable species, typically a dyestuff, is formed in an amount equivalent to the amount of substrate consumed in a fixed period of time, i.e., the substrate decreases with time to reach the endpoint of the enzyme reaction for a fixed period of time. Another method .is called the kinetic or rate-determining method for assaying enzyme activity which comprises measuring a detectable change of a physical amount with time; in this case, it is not required that the reaction be completed, unlike the endpoint method. More specifically, in the kinetic method a catalytic reaction proceeds due to interaction between the enzyme contained in a liquid sample and a substrate, and the amount of the reaction product increases or decreases with time in a linear relationship between the product produced and time. These two methods are explained in Method of Enzymatic Analysis, Hans Ulrich Bergmeyer, vol. 2, pages 726-773 (1974), published by Academic Press, Inc., New York, referring to measurement of transaminase activity. According to the Reitman-Frankel method (which is the endpoint method described in Amer. J. Clin. Pathol., vol. 28, page 56 (1956)), oxaloacetate produced after incubation for a fixed period of time is chemically converted into pyruvate; 2,4-dinitrophenylhydrazine is added to the pyruvate and the 2,4-dinitrophenylhydrazone formed is optically measured at 500 to 550 nm. On the other hand, according to the Karmen method (which is the kinetic or rate-determining method described in J. Clin. Invest., vol. 34, page 131 (1955)) as illustrated below, enzyme reaction (1') is immediately coupled with enzyme reaction (2'). In reaction (2'), wherein a rate of decreasing absorption of coenzyme NADH is measured at 340 nm. ##STR2##
In the past, the activities of transaminases have been measured by a color reaction in which a diazo dye is coupled with the pyruvate or oxaloacetate formed according to equations (1) or (2) (Japanese Patent Application (OPI) 40191/76) (the term "OPI" as used herein refers to a published unexamined patent application). The reaction system involved in the Japanese Patent Application supra belongs to the aforesaid endpoint method similar to the Reitmean-Frankel method. As an embodiment of this prior art method, a multilayer analysis film wherein the sequential reactions described above are intended to be separately conducted in two adjacent reagent layers has been proposed. The object is to reach equilibrium of the enzyme reaction of the first stage prior to beginning the coupling reaction of the second stage by conducting the two reactions at a suitable interval. Thus, this method runs counter to current needs in the art that reaction time (time required for assay) should be shortened as much as possible and, further, the sensitivity thereof is far from satisfactory.
There has also been proposed a method for the quantitative measurement of the activity of various transaminases where pyruvate is formed directly as shown in equation (1) above or indirectly from oxaloacetate formed according to equation (2) by way of a reaction in which another enzyme may or may not be involved, whereafter hydrogen peroxide - formed from the pyruvate by pyruvate oxidase (POP) according to equation (3)-, a color indicator composition and a peroxidase form a dye, according to equation (5). Thus, the resultant color level may be measured colorimetrically (for example, see Japanese Patent Application (OPI) 13068/80).
The measurement system of the present invention is a kinetic or rate-determining method similar to the Karmen method, in which applying several steps of conventional, enzymatically coupled reactions described hereinafter to reaction (1) or (2), the product produced (pyruvate in reaction (1) and oxaloacetate in (2)) is immediately converted into a detectable chemical species (NADH or a dye) and increase or decrease of the detectable chemical species is measured with time. In the rate-determining method utilizing several steps of coupled reactions, the enzyme activity of GOT or GPT should be reflected on the detectable chemical species to be actually measured.
The principle of the transminase assay used in the present invention which is described in Japanese Patent Application (OPI) 13068/80 is shown below. ##STR3## wherein: GOT: Glutamate-oxaloacetate transaminase
GPT: Glutamate-pyruvate transaminase PA1 Pi : Inorganic phosphoric acid PA1 POP: Pyruvate oxidase PA1 FAD: Flavin adenine dinucleotide PA1 TPP: Thiamine pyrophosphoric acid PA1 M.sup.2+ : Bivalent metal PA1 POD: Peroxidase PA1 (a) a pyruvate oxidase (hereafter often referred to as POP) is located in the analysis film where POP-catalyzed reaction (3) receives maximum exposure to oxygen in the air and therefore, POP is contained in the uppermost porous layer or an upper layer which is typically an enzyme layer located in contact with both the porous layer and the color indicator layer; and, PA1 (2)the impregnation amount of POP is from 1,000 to 100,000 U/m.sup.2.
As is evident from the reaction schemes above, GOT enzyme reaction (2) is coupled with reactions (4), (3) and (5) in turn and, GPT enzyme reaction (1) is coupled with reactions (3) and (5) in turn. Namely, pyruvate formed according to the GPT-catalyzed reaction (1) or formed by GOT-catalyzed reaction (2) coupled with reaction (4) by the action of oxaloacetate decarboxylase, is converted into hydrogen peroxide according to POP-catalyzed reaction (3) which is further coupled with indicator enzyme reaction (5).
The POD enzyme reaction (5) where the thus produced hydrogen peroxide is a substrate, effects a coupling reaction between a hydrogen donor and a coupler to form a dye which is then subjected to a colorimetrical quantitative measurement.
In the aforesaid coupled reaction system of a kinetic method, it is important that the enzyme activity of GOT or GPT be reflected on the detectable species (dye) actually measured. In other words, an intermediate product should not accumulate but once it is formed, it should be immediately converted into a final product. For example, when a rate of reaction (3) is slower than that of reaction (4), reaction (3) cannot proceed but pyruvate which is the product of reaction (4) simply accumulates. This is fatal to the rate-determining method since the GOT activity is not reflected on the dye in reaction (5). Contrary to the rate-determining method, no fatal problem occurs with the endpoint method because the reaction finally reaches the endpoint anyhow; merely a longer time is required to complete the reaction.
As described above, GOT or GPT activity is assayed through a series of chemically coupled reactions which are conducted in an aqueous solution. However, precise control of the weight or volume of the components and troublesome handling of an aqueous solution are needed in order to perform these complicated reactions at high efficiency. In addition, the analysis procedures require a long time. Accordingly, this is not a satisfactory method for clinical assays in which both quickness and accuracy are needed.
The normal concentration of transaminase in healthy human blood is about 20 IU/1 at most, and the quantity of the substrate on which the enzymatic action of transaminase occurs during several tens of minutes for the measurement according to the method of the prior art is on the degree of 10.sup.-4 mole/1 under optimum condition. In a method for the colorimetric measurement of such trace amounts of a substrate using a coupled enzyme reaction, it is required--to maintain the accuracy of the results and to shorten the time for the analysis procedure--that: (1) each reaction fully proceed; (2) the dye formed have high absorbance; (3) the dye remain stable until it is photometrically measured; and (4) the dye formed not be lost by migration or diffusion prior to measurement in the case of a colorimetrical measurement using a film for quantitative analysis.
It is known that various oxidase enzymes, e.g., lactate oxidase or cholesterol oxidase, can be incorporated in a reagent layer or a spreading layer (see U.S. Pat. Nos. 4,166,763 issued to Esders et al and U.S. Pat. No. 3,983,005). However, the methods disclosed in these patents relate to endpoint colorimetry in which a dye is formed in an amount equivalent to substrate in a fixed period of time-, i.e., the dye is measured at the endpoint of the reaction. In these methods, there is no reason to give consideration to a rate-determining step. It is thus sufficient, as taught in U.S. Pat. No. 4,166,763, that 500 U/m.sup.2 of oxidase enzymes be present at maximum in a slide for the endpoint method. In view of these points, the present inventors have conducted extensive investigations and have found that the feed of oxygen, one of the substrates for the pyruvate oxidase, is a rate-determining step in the successive reaction system described above, and that the rate at which the hydrogen peroxide migrates from one layer of the multilayer film to another is much higher than that of migration rates of other substrates or products; the present invention has thus been accomplished.